Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification. Here, we outline a series of methods that employ transient reporters of editing enrichment (TREE) to facilitate the highly efficient single-base editing of human cells at precise genomic loci. Briefly, these transient reporters of editing enrichment based methods employ a transient episomal fluorescent reporter that allows for the real-time, flow-cytometry-based enrichment of cells that have had single nucleotide changes at precise genomic locations. This protocol details how these approaches can enable the rapid (~3–4 weeks) and efficient (clonal editing efficiencies >80%) generation of biallelic or multiplexed edited isogenic hPSC lines using adenosine and cytosine base editors.

脱氨酶融合 Cas9 碱基编辑技术实现了精确的单核苷酸基因组编辑，无需引入破坏性双链断裂和低效的同源定向修复。然而，目前分离碱基编辑细胞群的方法是无效的，特别是当与人类多能干细胞（一种对基因组修饰具有抵抗力的细胞类型）一起使用时。在这里，我们概述了一系列使用编辑富集瞬时报告基因（TREE）的方法，以促进人类细胞在精确基因组位点的高效单碱基编辑。简而言之，这些基于编辑富集方法的瞬时报告基因采用瞬时附加型荧光报告基因，可以对在精确基因组位置发生单核苷酸变化的细胞进行实时、基于流式细胞术的富集。该协议详细介绍了这些方法如何使用腺苷和胞嘧啶碱基编辑器快速（约 3-4 周）和高效（克隆编辑效率 >80%）生成双等位基因或多路编辑的同基因 hPSC 系。